Human cytomegalovirus (CMV) infection of polarized cells is central to invasion at the portal of entry and virus dissemination in the host. This applicant's hypothesis is that key CMV envelope glycoproteins, gB and the gH complex, contain sorting signals recognized by cognate partners in the endocytic pathway that are co-opted to direct virion envelopment and egress from a cytosolic compartment. CMV gB, the major component of the virion envelope, is transported to the apical membrane domain where virions egress in polarized human epithelial cells. In transfected cells, gH targets to basolateral membranes. Both glycoproteins traffic in the endocytic pathway. Our unexpected new finding, that gB and the gH complex converge in a perinuclear endocytic compartment with virion matrix proteins, establishes that envelopment occurs in cytosolic vesicles of epithelial cells and suggests that virion egress could be regulated by glycoproteins sorted in common endosomes and/or the apical recycling compartment. A central question is how the sorting signal hierarchy in gB and gH complex modulates virion envelopment and egress routes in the context of two polarized cell types: epithelial and endothelial cells. The specific aims are to: (1) Finish identifying functional sorting signals in CMV gB and gH complex used for trafficking in polarized cells. The hypothesis is that the cytosolic domains of these glycoproteins contain multipartite signals that regulate their trafficking in endocytic pathways. (2) Continue to identify cognate binding partners co-opted by cytosolic sorting signals in CMV gB and gH complex. The goal is to determine which cellular proteins regulate their transport in the endocytic/recycling pathway. (3) Elucidate CMV virion envelopment and egress routes in the context of infected polarized epithelial cells and the signal hierarchy that modulates this pathway. (4) Investigate CMV glycoprotein sorting and virion envelopment and egress pathways in endothelial cells, another polarized cell type that plays a critical role in CMV infection. These experiments will advance knowledge of transport motifs and cellular molecules that modulate virion envelopment and egress routes in polarized epithelial cells and improve understanding of virus release from endothelial cells as a model for transmission from vascular beds in vivo. Conceivably, these findings will be of interest to cell biologists and could have medical relevance to vascular biology and organ transplantation. Possibly, the strategies used by CMV for selective targeting to endocytic subcompartments could be incorporated into novel approaches for introducing antiviral compounds into cytosolic vesicles in polarized cells in order to preclude virion envelopment and egress.